\chapter{Protocol}





\section{Cell culture}
\label{sec:protocol-cell-culture}

\subsection{Starting hood}

\begin{enumerate}
\item Turn on UV lights for 20 minutes.
\item	Turn off UV lights.
\item	Press Start and Lights button.
\item	Wait for hood to start (air flow meter in the green; if alarm sounds press alarm button).
\item	Spray work surface of hood with 70 \% ethanol and allow to dry.
\end{enumerate}

\subsection{Thawing and plating cells}
\begin{enumerate}
\item Pre-equilibrate 10 ml DMEM/10\% CS (see Recipie \ref{recp:DMEM10CS}) in a 10 cm dish in incubator at 37 C /10\% CO2.
\item	 Thaw an ampule rapidly by immersion in a water bath at 37C.
\item Wipe off the outside and rinse with 70\% ethanol.
\item	 Remove the cells with a 1 ml pipette and add to the dish.
\item	 Rock the dish to disperse any clumps.
\item	 Follow the cells to confluence under the microscope. Confluence will take 2 to 4 days, and the cells should be fed on day 2 after plating. \\ Note: There is vial-to-vial variation in growth rates. If a vial does not grow well, thaw another.
\end{enumerate}

\subsection{Hood shutdown}
\begin{enumerate}
\item	Turn off airflow and lights.
\item	Clean work surface of hood with approved cleaner (spray and wipe dry).
\item	Record hood use in book
\end{enumerate}



\section{Splitting cell culture}
\label{sec:protocol-splitting-cell-culture}

This should be done when the cells reach confluence.  No spaces between cells, but before cells appear to look crowded.  This is the time to trypsinize and replate for both carry-ons and ones to be differentiated. If replated too soon, the time required to reach confluence for differentiation is longer. If replated after confluence, the cells do not release from the dish as well and do not grow as fast when replated.

\subsection{Starting Hood}

\begin{enumerate}
\item Turn on \ac{UV} lights for 20 minutes.
\item	 Turn off \ac{UV} lights.
\item Press ``Start' and ``Lights'' button.
\item Wait for hood to start (air flow meter in the green; if alarm sounds press alarm button).
\item Spray work surface of hood with 70 \% ethanol and allow to dry.
\end{enumerate}


\subsection{Trypsinization Protocol}

\begin{enumerate}
\item Wash cells 1 x with 10 ml \ac{DMEM} for each 10 cm dish.
\item Aspirate \ac{DMEM}
\item Apply 2.5 ml 0.05 \% trypsin solution (a note says it is possible to use the 0.25 \% stock) in \ac{PBS} at room temperature.
\item	Allow trypsin to remain on cells for a few minutes (2-3 min) depending on the age of the trypsin solution - the newer the solution, the less time it will take to lift the cells - until the monolayer turns opaque.
\item	Triturate the cells with a 5 ml pipette, until they have all been removed from the dish.
\item	Dilute the trypsin-cell suspension in the volume of medium required to plate.  Ratio for splitting is 1/18, and so the cells are put into 180 ml of DMEM/10\% CS (see Recipie \ref{recp:DMEM10CS}).
\end{enumerate}

Since the thawed cells are usually at passage 6 each week the cells gain 2 passages. After about passage 11 (3 weeks) the cells do not differentiate as consistently, so start a new vial.

\subsection{Hood shutdown}
\begin{enumerate}
\item Turn off airflow and lights.
\item	Clean work surface of hood with approved cleaner (spray and wipe dry).
\item	Record hood use in book
\end{enumerate}

\section{3T3-L1 Differentiation}
\label{sec:differentiation}

The timing of differentiation protocol is important. If it is begun too soon after confluence, or too late after confluence, differentiation is less complete.  The lawn of cells has a smooth opaque appearance at about day 3-4 after differentiation has begun. Under the scope you will see fat droplets. A poorly differentiated plate will appear "splotchy"  i.e. patches of clearer fibroblast among patches of opaque fat cells. As the time goes by, the fat droplets increase in size and number.

During the time the cells are undergoing the phenotypic change from fibroblasts to adipocytes they are not attached very securely to the tissue culture dish. Be very carefu1 when removing the differentiation medium otherwise many cells will be aspirated with the medium. Fresh medium should be added carefully at this stage (avoid vigorous squirting of medium). These precautions also apply to the insulin medium, although it is not as crucial. To avoid releasing cells, tilt the plates when aspirating spent medium and adding fresh medium. Put a Styrofoam test tube rack under the stainless steel incubator tray holding 35 mm dishes, or a magic marker directly under a 4-pile of 10 cm plates.

With an occasional lot of serum, the cells are very metabolically active during differentiation and the medium becomes acid (yellow) in 48 h. If this happens during the exposure to the differentiation medium or in the next feeding (insulin alone), some cells may die or be released. Generally exposure for 48 h to these media will work, but if the medium becomes very acid sooner, it is necessary to replace the medium with DMEM/FBS/INS sooner (or with DMEM/FBS). It is best to apply the differentiation medium and the insulin medium in the afternoon, so that an earlier feeding can be done on day 2 if necessary.

The cells are normally used at day 8 to day 12 post-differentiation (taking day of addition of differentiation medium as day 0).



\subsection{Starting hood}
\begin{enumerate}
\item Turn on \ac{UV} lights for 20 minutes.
\item	Turn off \ac{UV} lights.
\item	Press ``Start'' and ``Lights'' button.
\item Wait for hood to start (air flow meter in the green;  if alarm sounds press alarm button).
\item	Spray work surface of hood with 70\% ethanol and allow to dry.
\end{enumerate}

\subsection{Differentiation}
\begin{enumerate}
\item	Let these cells reach confluence (about 3-4 days for l/20 split), and then go 2 days further.
\item	Feed every 2 days with \ac{DMEM}/10\% \ac{CS} (see Recipie \ref{recp:DMEM10CS}).
\item	Start the differentiation protocol by replacing the \ac{DMEM}/10 \% \ac{CS} with Differentiation Medium (see Recipie \ref{sec:dex})
\item	After 48 hours replace the Differentiation Medium (see Recipie \ref{sec:dex}) with Insulin Medium (see Recipie \ref{sec:insulin-medium}).
\item	Grow the cells for a further 48 hours and then replace the medium with Adipocyte Medium (see Recipie \ref{recp:adipopcyte-medium})
\item	Change the medium every 2-3 days.
\end{enumerate}

\subsection{Hood shutdown}
\begin{enumerate}
\item Turn off airflow and lights.
\item	Clean work surface of hood with approved cleaner (spray and wipe dry).
\item	Record hood use in book
\end{enumerate}



\section{Media preparation}

The bottles and plastic test tubes put into the hood are sprayed with 70 \% ethanol and allowed to dry outside the hood.  At the beginning, the openings of bottles and tubes to be used are flamed. This can be done with plastic tube if it is done briefly. The serum is frozen in 25 ml lots and is poured directly into the DMEM. Media is made up for a week and dispensed into separate bottles for the several feedings.  An exception to this case is the differentiation medium, which must be made up within 2 days of use.

\section{Snap freezing of cell culture}
\label{sec:snap-freezing}
Snap freezing is the rapid freezing of cell culture in liquid Nitrogen according to the following protocol.

\begin{enumerate}
 \item Aspirate Medium
\item Wash 2X with 10 ml \ac{DMEM} (see section \ref{sec:dmem})
\item Wash 2X with 10 ml \ac{PBS} (see reagent \ref{recp:pbs})
\item Add 1ml of Complete Homogenisation Buffer (see section \ref{sec:complete-homog-buffer})
\item Scrape plate with sterile cell scraper
\item Pass 5X through 3ml syringe with 23G 1 1/4 needle
 \item Replace into yellow eppendorf.
 \item Snap Freeze in Liquid Nitrogen.
 \item Freeze at –30.
\end{enumerate}


\section{TNF-alpha Induced Insulin resistance}
\label{sc:tnf-induced}

Fully differentiated 3T3-L1 adipocytes treated with 3ng/ml recombinant murine \ac{TNF} to induce insulin resistance. This protocol involves both a control set and a treated set of cultures cells.

\subsection{Control (insulin-sensitive) cells}
\label{sec:tnf-control-protocol}

\begin{enumerate}
 \item Aspirate medium
 \item Add 10 ml Adipocyte Medium.(see section \ref{recp:adipopcyte-medium})
 \item Replace with fresh Adipocyte Medium (see section \ref{recp:adipopcyte-medium}) every 24 hours for three days.
\end{enumerate}



\subsection{TNF-alpha induced insulin resistance}
\label{sec:tnf-exposure-protocol}

\begin{enumerate}
 \item Aspirate medium
  \item Add 10ml \ac{TNF}/Adipocyte Medium (see section \ref{recp:tnf-adipo-medium}).
  \item Replace with fresh \ac{TNF}/Adipocyte Medium every 24 hours for three days.
\end{enumerate}

\subsection{References}

Protocol adapted from: \cite{Sartipy2003}.

\section{Insulin-induced insulin resistance of Cultured cells}
\label{sec:insulin-induced}

Fully differentiated 3T3-L1 adipocytes treated with chronic levels of insulin to induce insulin resistance. This protocol involves both a control set and a treated set of cultures cells.

\subsection{Control (insulin sensitive) cells}
\begin{enumerate}
 \item Incubated overnight (12-14hrs) in Adipocyte Medium (see section \ref{recp:tnf-adipo-medium})
\end{enumerate}


\subsection{Insulin-induced insulin resistance}

\begin{enumerate}
 \item Adipocytes were treated with Adipocyte Medium/10nM Insulin (see Section \ref{recp:adipo-insulin})
  \item Incubated overnight (12-14hrs)
\end{enumerate}


Overnight Incubations were limited to periods of 12-14 hours to minimise complications due to effects of glucose deprivation on glucose transport.

Overnight induction of insulin resistance

\subsection{Pre-treatment}

\begin{enumerate}
 \item 3T3 adipocytes treated with Adipocyte Medium (see section \ref{recp:adipopcyte-medium}) for 48hrs prior to inducing insulin resistance
\end{enumerate}

\subsection{References}

Protocol adapted from \cite{Ross2000}.

\section{Subcellular fractionation of 3T3-L1 adipocytes}

Method adapted from \cite{Simpson1983}.

\subsection{Preparation before starting}

Make up 500 ml \ac{HES} buffer at pH 7.4 (See Recipie \ref{recp:hes}). This is stable at 4 $^\circ$C but protease inhibitors (See Section \ref{recp:prot-inhib}) need to be added to the HES buffer fresh on the day of the subfractionation. Take the volume of HES that will be required for the subfractionation. Place on ice and add the appropriate volume of inhibitor (these are aliquoted out and stored at -20 $^\circ$C). The final concentrations of inhibitors are pepstatin A 1$\mu$g/ml, E64 20$\mu$M and aprotinin 50$\mu$M. Take care to label all centrifuge tubes `- insulin' or `+ insulin' and keep all tubes on ice or at 4 $^\circ$C throughout the subfractionation.


\subsection{Subfractionation protocol}


\begin{enumerate}
 \item Wash all plates of cells with 2 x 5 ml serum free media (Reagent \ref{sec:dmem}) then incubate in 10 ml of the same media for two hours.
 \item For insulin stimulated cells, add 10 nM insulin (Reagent \ref{recp:insulin}) 20 minutes prior to harvesting (and change the serum free media on the others (Reagent \ref{sec:dmem})).
 \item Remove medium from cells.  Wash each plate with 2 x 5 ml PBS (Reagent \ref{recp:pbs}).
 \item Add 4 ml \ac{HES} (Recipie \ref{recp:hes}) buffer per plate. Scrape the cells from the plate using a cell scraper. *Do any insulin stimulated cells first as the timing is more important with these.* 
 \item Wash the plates again with 1ml HES (Recipie \ref{recp:hes}) and transfer to the next plate, then add to the homogeniser.
 \item Transfer the cells to the homogeniser and homogenise the cells by hand using 10 strokes of a Teflon/glass homogeniser.
 \item Keep 100$\mu$l of the homogenate for analysis later and centrifuge the rest at 19,000g for 20 min at 4$^\circ$C (12,500rpm) in a Beckman centrifuge (JA-20 rotor). Balance the tubes before centrifugation by weighing and topping up with HES (Recipie \ref{recp:hes}) if necessary.  
 \item The supernatant obtained from this spin is used to prepare intracellular fractions (See Section \ref{sec:intracell-fraction}) and the pellet is retained on ice for the preparation of plasma membranes (See Section \ref{sec:plasma-fraction}).
\end{enumerate}




\subsubsection{Preparation of intracellular fractions}
\label{sec:intracell-fraction}

\begin{enumerate}
 \item The supernatant from the first spin is recentrifuged at 40000g for 20 min at 4$^\circ$C in the JA-20 rotor in a Beckman centrifuge. (This is 18.25Krpm). This yields a pellet of \ac{HDM}. The pellet can be retained on ice while the next spin is being organised.

\item Transfer the supernatant to Beckman Ultracentrifuge tubes. Top up, balance and cap the tubes (insert and cap). Spin at 180000g (43Krpm) for 75 min at 4$^\circ$C in a Ti-80 rotor in the Ultracentrifuge. This needs 40Krpm.This spin yields a pellet of \ac{LDM}.

\item Keep the supernatant from this spin (labelled cytosol) and resuspend the pellet in a small volume of \ac{HES} buffer (~0.5 ml) using a small homogeniser. Aliquot, freeze and store as described in Section \ref{sec:freeze-store}. Note that the \ac{LDM} pellet is quite small and fragile so aspirate the supernatant carefully in order to avoid disrupting the pellet and losing material. The \ac{HDM} pellet should be dealt with in a similar manner 

\end{enumerate}

\subsubsection{Preparation of plasma membranes}
\label{sec:plasma-fraction}


\begin{enumerate}
\item Each pellet from the first spin is resuspended (using a 1 ml pipette then in the homogeniser) in 8 ml \ac{HES} buffer and layered on to 4 ml of 1.12 M sucrose in HES (Recipe \ref{recp:sucrose-hes}). The total tube volume is 12 ml. 

\item Insert the balanced tubes into the SW-40 rotor and centrifuge at 100,000g for 60 min at 4$^\circ$C in the Ultracentrifuge (36,000rpm in the SW-40).  Note that all buckets have to be placed in the correct positions in the rotor and spun whether or not they contain centrifuge tubes.

\item This spin yields plasma membranes as a fluffy layer at the interface between the sucrose layers. There is also a pellet and a thin layer of free fat collects on the surface of the \ac{HES}. Collect the plasma membranes by careful aspiration using a Pasteur pipette and transfer them into a polycarbonate JA-20 tube. The pellet can retained if required (labelled \ac{M/N}) and prepared for storage Section \ref{sec:freeze-store}.

\item Make the volume up with fresh \ac{HES} to fill the tubes and dilute out any concentrated sucrose that has been transferred. Pellet the plasma membranes by centrifugation at 40,000g for 20 min at 4$^\circ$C in the JA-20 rotor in a Beckman centrifuge. (This needs 18,250rpm).  

\item Discard the supernatant and resuspend the pellet in a small volume of \ac{HES} and prepared for storage Section \ref{sec:freeze-store}

\end{enumerate}

\subsubsection{Aliquot, Freeze and Store}
\label{sec:freeze-store}

Aliquot it into Eppendorf tubes and snap freeze in liquid nitrogen. Retain a portion for protein assay and store all preps at -80$^\circ$C until use. (Note that the buffer chosen in which to resuspend the plasma membranes and other preps will depend on what work is planned for them e.g. Hepes interferes with some protein assays).

% \begin{figure}
%  \centering
%  \includegraphics[scale=0.5]{lab-book-images/SubcellularFractionation.png}
%  % SubcellularFractionation.png: -1238670960x-1238695792 pixel, 0dpi, 1232004255318016.00x1232028951379968.00 cm, bb=
%  \caption{Subcellular Fractionation}
%  \label{fig:subcellular-fractionation}
% \end{figure}


\section{Protein quantification by Lowry Procedure}
\label{sec:lowry}

\begin{enumerate}
 \item In the appropriately labelled plastic microcentrifuge tubes, dilute the stock protein standard solution in a total volume of 1 ml.  The standard solutions are shown below in Table


\begin{table} [ht]
\caption[Lowry Protein Standards]{Lowry Protein Standards}
\begin{center}

\begin{tabular}{| p{3cm} |p{3cm} |p{3cm} |p{3cm} |}
\hline
  Volume of 400 $\mu$g/ml protein standard solution ($\mu$l) & Volume of dieonised water ($\mu$l) & Protein concentration ($\mu$g/ml) \\
\hline
	0 & 1000 & 0 \\
	25 & 975 & 10 \\
	50 & 950 & 20 \\
	100 & 910 & 40 \\
	150 & 850 & 60 \\
	200 & 800 & 80 \\
	250 & 750 & 100 \\
	375 & 625 & 150 \\
	500 & 500 & 200 \\
\hline
\end{tabular}
\end{center}
\end{table}


\item Label the microcentrifuge tube containing the protein concentration 0$\mu$g/ml as the ‘BLANK’.
\item Put the sample(s) in microcentrifuge tube(s) and label.  Again, dilute to a total volume of 1 ml
\item Add 100$\mu$l of the \ac{DOC} solution to each of the three types of tube (STD, BLANK and SAMPLE)
\item Vortex mix then leave for 10 minutes at room temperature.
\item Add 100$\mu$l \ac{TCA} to all tubes and vortex mix.
\item Pellet the precipitates by centrifugation for 10 minutes at max revs.
\item Decant and blot away the supernatants.
\item Add 1 ml of Lowry modified reagent to each tube then allow the pellets to dissolve. 
\item Transfer the solutions to their correspondingly labelled test tubes.  
\item Rinse each microcentrifuge tube with 1ml of water and add the rinsings to their respective test tubes.  
\item Ensure the contents of the tubes are mixed well and leave the solutions to stand for 20 minutes at room temperature.  
\item With rapid and immediate mixing, add 0.5ml Folin and Ciocalteu’s Phenol reagent to each tube.
\item Leave tubes to stand at room temperature for 30 minutes.  Purple/blue colour should now develop. 
\item At this stage turn on spectrophotometer, to allow it to warm up. 
 \item Transfer the solutions to cuvettes for measurement of absorbence at a wavelength between 500nm and 800nm (usually 700nm). 
\item Measure the absorbence of the STD and SAMPLES against the BLANK.  Readings should be completed within 30 minutes.  
\item Create a calibration curve by plotting the absorbence of the STDs against their known concentrations.
\item From this calibration curve calculate the concentration of protein in the SAMPLE tube(s).  This is done by multiplying the results by the dilution factor to obtain the protein concentration in the original sample.
\end{enumerate}




